Blockage of the lysosomal system that degrades unwanted protein has emerged as a common theme in neurodegenerative diseases. At "Parkinson’s Disease: Genetics, Mechanisms, and Therapeutics," a meeting held 2-7 March in Keystone, Colorado, researchers proposed ways to revive that process, including kicking protein trafficking to the lysosome into high gear. The meeting built on the connection between the toxicity of α-synuclein, a hallmark of Parkinson's pathology, and GBA1, a genetic risk factor for the disease. GBA encodes the lysosomal lipid-degrading enzyme glucocerebrosidase (GCase). At Keystone, researchers reported that α-synuclein blocks folding and trafficking of the enzyme to that organelle. They presented small-molecule chaperones that can refold GCase and deliver it to lysosomes, where it could potentially benefit PD patients.
“If you have a version of GCase that’s not folded properly but is still made in the neuron, then the refolding idea is an attractive one,” commented Mark Cookson of the National Institutes of Health. “GBA1 is a genuine PD risk factor for significant numbers of people, so even if you could only treat GBA1-mutation-associated PD, that would be a huge hit.” Because α-synuclein inclusions play a role in other neurodegenerative diseases as well, Keystone attendees proposed that the therapy could be more broadly applied, perhaps even to Alzheimer's.
Mutations in GBA1 also cause Gaucher’s disease (GD), which is characterized by a buildup of lipids in lysosomes. Some forms of GD cause neurodegeneration in the brain (see Jan 2014 news story). The homozygous mutations that trigger GD are rare, but heterozygous mutations in GBA1 are common and increase a person’s chances of developing Parkinson’s disease and dementia with Lewy bodies (see Apr 2013 news story and PDGene).
How GBA1 mutations influence these synucleinopathies remains a hot topic. In 2011, researchers from the lab of Dimitri Krainc, then at Massachusetts General Hospital, Boston, proposed that GCase and α-synuclein form a destructive feedback loop in which the accumulation of the GCase substrate glucosylceramide stabilizes α-synuclein oligomers, which in turn inhibit the activity of the enzyme.
At Keystone, Krainc, now at Northwestern University, Chicago, suggested that the toxic loop may roll in reverse: Accumulation of α-synuclein can dampen GCase activity, increasing glucosylceramide. To arrive at this conclusion, Joseph Mazzulli, who was a postdoc with Krainc but now runs his own lab at Northwestern, nurtured midbrain neurons derived from induced pluripotent stem cells (iPSCs) made from a PD patient who carried two extra copies of the a-synuclein gene. Mazzulli grew the neurons for more than a year, a feat that awed researchers at the meeting (see image below). As the cells aged, the neurons with the α-synuclein triplication started ramping up production of lysosomes. However, after about 200 days in culture, “that compensation started to fail,” Krainc said. Despite the increase in lysosomal biogenesis, α-synuclein gradually accumulated in the cells, and the level of the protein was “striking” by day 200, he said. After 420 days the cells were still viable, however, the activity of GCase was dramatically lower than in neurons derived from normal iPSCs. The researchers observed an accumulation of GCase in the endoplasmic reticulum (ER) fraction, suggesting that trafficking of the enzyme from the ER through the Golgi was blocked. Interestingly, the α-synuclein accumulation also seemed to hold up other enzymes that traffic through the Golgi pathway, such as β-galactosidase.
Krainc reasoned that “opening up lanes” in the ER-to-Golgi pathway might relieve the congestion. Indeed, when the researchers overexpressed Rab1a, a GTPase known to promote ER-to-Golgi trafficking, α-synuclein levels in the neurons dropped, and GCase activity was restored. The researchers achieved a similar result by dosing the cells with a small-molecule chaperone of GCase (Rogers et al., 2013). The molecule helps GCase fold properly and ferries the enzyme through the vesicular pathway into the lysosome, where it can do its job, Krainc said. Because the chaperone approach works to enhance the function of both wild-type and mutated forms of GCase, companies are pursuing similar molecules as potential therapies for PD and GD.
Several researchers in the audience praised the work, but some questioned whether this approach could be applied to patients with sporadic PD, who do not all have the same amount of α-synuclein accumulation. Others wondered whether the approach could work for other neurodegenerative diseases marked by protein toxicity. Krainc cautioned that the GBA1 target was, so far, mechanistically linked only to α-synuclein accumulation.
“It is very hard to know how well the mechanisms will generalize,” Cookson told Alzforum. “But α-synuclein has always been thought of as a difficult target, so maybe targeting GBA1 is a way into α-synuclein biology and α-synuclein-based therapy.”
Pfizer is one company interested in targeting PD through GBA1. Zdenek Berger from the company's Cambridge, Massachusetts, lab reported that compounds identified in the same screen that Krainc used for his studies activated GCase in human brain extracts. Pfizer is pursuing GCase as a target for several neurologic diseases, Berger told Alzforum.
Patrick Lewis of the University of Reading in England commented that while the link between GBA1 and synucleinopathies is gaining traction, it is important to remember that not everyone with a GBA1 mutation develops PD. “It’s a risk factor, but not a directly causative mutation,” Lewis said. “That makes the translation more difficult.” Nevertheless, Lewis acknowledged that another genetic link strongly implicates lysosomal dysfunction in PD. Mutations in the lysosomal transport gene ATP13A2 lead to early onset parkinsonism and α-synuclein accumulation (see Usenovic et al., 2012, and Tsunemi et al., 2013).
The Keystone meeting featured more data that meshed with α-synuclein’s ability to waylay GCase in the ER. For example, Vikram Khurana of the Whitehead Institute for Biomedical Research in Cambridge, Massachusetts, suggested that ramping up ER-associated degradation (ERAD), which disposes of misfolded proteins by trafficking them out of the ER, can overcome α-synuclein toxicity as well. Khurana’s recent yeast screen identified Hrd1, an E3 ubiquitin ligase that promotes ERAD, as a suppressor of α-synuclein toxicity. Overexpressing Hrd1 in iPSC-derived neurons from a PD patient with the A53T α-synuclein mutation reduced both α-synuclein accumulation and the retention of GCase in the ER (see Oct 2013 news story). Further, Khurana reported reduced α-synuclein toxicity and less build-up of GCase in the ER as a result of treatment with N-aryl benzimidazole (known as NAB2), a small molecule identified in a yeast screen as a promoter of endosomal trafficking, These results resemble Krainc’s on Rab1a overexpression or treatment with GCase chaperones. Overall, researchers from several labs presented a handful of approaches using different ways of increasing ER-mediated or lysosomally mediated protein degradation, all of which alleviated cellular phenotoypes of PD pathology.
“If the trafficking of GBA1 is perturbed by α-synuclein, then we’re hoping that NAB2 could be a compound that would reverse that trafficking defect,” Khurana said. “Because α-synuclein may perturb trafficking of many different kinds of protein, a compound like NAB2 might be able to rescue multiple defects at once.”
Khurana will conduct more comprehensive yeast screens to identify genes that rescue or exacerbate α-synuclein toxicity either alone or together. From that, he plans to build genetic interaction maps that could help probe human iPSC-derived neurons for what he calls “druggable nodes,” or pathways implicated in disease pathology.
Besides GCase, Khurana also found that nicastrin, a component of γ-secretase, accumulates in the ER of PD neurons. Whether this connects AD to synucleinopathy remains to be shown, but it does hint that protein trafficking defects may play a broader role in neurodegenerative disease, said Khurana.
Along that vein, Hui Zheng of Baylor College of Medicine in Houston implicated a transcription factor that controls autophagy in cleaning up another AD pathology. Zheng found that overexpression of the factor reduced the presence of neurofibrillary tangles and rescued neurodegenerative defects, including neuronal loss, deficits in synaptic plasticity, and declines in learning and memory as measured by the Morris water-maze challenge, in Tg4510 mice expressing a mutant human tau gene. Zheng, who co-organized the conference, suggested that the factor clears tau via a two-pronged approach: by directly turning on lysosomal genes, and by boosting the expression of PTEN, a protein that promotes autophagy.
Said Khurana, “Lysosomal dysfunction has emerged as a theme encompassing different neurodegenerative diseases, but the mechanisms that perturb endosomal trafficking and lysosomal function may be highly distinct between the diseases.”—Jessica Shugart
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Research Models Citations
- Usenovic M, Knight AL, Ray A, Wong V, Brown KR, Caldwell GA, Caldwell KA, Stagljar I, Krainc D. Identification of novel ATP13A2 interactors and their role in α-synuclein misfolding and toxicity. Hum Mol Genet. 2012 Sep 1;21(17):3785-94. PubMed.
- Tsunemi T, Krainc D. Zn²⁺ dyshomeostasis caused by loss of ATP13A2/PARK9 leads to lysosomal dysfunction and alpha-synuclein accumulation. Hum Mol Genet. 2014 Jun 1;23(11):2791-801. Epub 2013 Dec 13 PubMed.